Novel direct detection method for quantitative determination of intracellular nucleoside triphosphates using weak...

A novel analytical method has been developed for direct quantification of intracellular nucleoside triphosphates (NTPs). Lysates of human peripheral blood mononuclear cells (PBMCs) were extracted by protein precipitation, and the filtered extracts were analyzed by weak anion exchange liquid chromatography (WAX-LC) coupled to detection by mass spectrometry (MS). Compared with ion pairing (IP)-LC/MS/MS, the only MS-compatible direct detection method for NTPs currently available, the new method completely avoids the usage of ion-pairing reagents and has a shorter analytical time of only 2 min. The method was validated and is being used to determine the amount of the triphosphate metabolite of D-D4FC (DPC817), an investigational HIV nucleoside reverse transcriptase inhibitor (NRTI), in human PBMC samples from clinical studies. By using a PE Sciex API 4000 triple quadrupole instrument operating in positive ion MRM mode, the method was able to achieve a lower limit of quantitation (LLOQ) of 5 fmol/10(6) cells in samples containing 3 x 10(6) lysed cells (6 fmol on-column). With minor adaptation, the method described here may be suitable for analyzing other NTPs. This paper also provides a discussion of the unique retention characteristics of WAX-LC, the principles of which may prove to be valuable for designing other forms of directly coupled ion-exchange (IX)-LC/MS methods suited for high sensitivity quantitative analysis.